Real-Time PCR Targeting Mosaic penA XXXIV for Prediction of Extended-Spectrum Cephalosporins Susceptibility in Clinical Neisseria Gonorrheae Isolates

BACKGROUND: Antimicrobial-resistant Neisseria gonorrheae (NG) is a global public health problem, resulting in limited empirical treatment options. Due to increasing minimum inhibitory concentrations (MICs) of ESCs against NG in the US, it is critical that susceptibility to ESCs be monitored. Since few laboratories routinely perform culture and susceptibility testing for NG, there is a need for a rapid test to predict susceptibility to ESCs. More than 98% of isolates with decreased susceptibility to cefixime (CFM) in the US carry mosaic penA XXXIV. In this study, we developed a multiplex real-time PCR for mosaic penA XXXIV and previously validated gyrA to predict ESCs MICs and ciprofloxacin (CIP) susceptibility. METHODS: 150 NG isolates with known cefpodoxime (CPD), CFM, ceftriaxone (CRO) and CIP MICs were obtained from Neisseria Reference Laboratory at University of Washington and CDC Antimicrobial Resistance Bank. DNA extracted from culture was used in multiplex HybProbe real-time PCR on Lightcycler 480. gyrA was genotyped by melt curve and served as internal control, while presence of mosaic penA XXXIV was detected by selective amplification. RESULTS: All 32 (100%) CIP-susceptible and 118 (100%) CIP-resistant isolates, as determined by Clinical and Laboratory Standards Institute breakpoints, demonstrated wild-type and Ser91 mutant gyrA genotype, respectively. Melt curve genotyping demonstrated mosaic penA XXXIV melt patterns in 66/68 (97%) isolates with at least one ESC MIC above alert value set forth by the CDC (CPD and CFM MICs ≥0.25 µg/ml; CRO MIC ≥0.125), while all 82 (100%) isolates with ESC MICs under alert values did not amplify. The first of the 2 false-negative isolates had MICs above alert values for all ESCs tested and harbored IX mosaic type, while the second one had CRO MIC above alert value and harbored XII mosaic type. Both of these mosaic types did not share homology with mosaic penA XXXIV in the region targeted by the assay. CONCLUSION: The mosaic penA XXXIV assay demonstrated 97% sensitivity and 100% specificity in predicting alert ESCs MIC values among clinical isolates tested, and was successfully multiplexed with gyrA assay. Clinical utility of this assay may be limited due to false negativity in isolates with non-XXXIV mosaic types, but it could serve as a useful surveillance tool for XXXIV mosaic. DISCLOSURE: R. Humphries, Roche: Consultant, Consulting fee